The present invention is related to the purification of plasmid and other DNA.
The purification of plasmid DNA from bacterial lysates is a rate-limiting and time-consuming step in molecular biology. The preparation of plasmid DNA for cloning and other purposes generally follows the scheme established in Birnboim (1983, Methods in Enzymology 100:243-255) in which the cleared bacterial lysate is applied to a cesium chloride gradient and centrifuged for 4-24 hours. This is usually followed by the extraction and precipitation of the DNA to yield DNA that is sometimes, but not always, free of RNA, protein and chromosomal DNA. Other methods employing cleared lysates to prepare DNA of similar quality are ion exchange (Colpan et al., 1984, J. Chromatog. 296:339-353) and gel-filtration (Moreau et al., 1987, Analyt. Biochem. 166:188-193) high-performance methods. While these latter methods generally work well as alternatives for ScCl gradients, they require costly solvent delivery systems and the reprecipitation of the isolated DNA fractions since they usually contain salt or are too dilute, and are limited in the amount of DNA that can be prepared (&lt;500 .mu.g) per run. Since typically 1 liter cultures of E. coli yield &gt;2 mg of plasmid DNA plus much RNA and protein, the capacity of the high performance methods requires multiple cycles to process these quantities of DNA.
It was discovered by Marko et al. (1981, Analyt. Biochem. 121:382-387) and Vogelstein et al. (1979, Proc. Nat. Acad. Sci. 76:615-619) that when DNA-containing extracts were applied in high concentrations of sodium iodide or sodium perchlorate, the DNA alone will bind to ground scintillation vials or ground GF/C glass fiber disks. RNA and protein do not bind. The bound DNA will elute eventually in water. It was also reported that DNA does not bind to and thus cannot be purified using other fine silicas such as silica gel and porous glass beads (Vogelstein, supra). A product, GENECLEAN.TM. (Bio 101, La Jolla, Calif.), is now commercially available for the purification of DNA which includes ground glass slurry, saturated NaI and 50% ethanol wash buffer.
Diatomaceous earth has been used for filtration, in chromatography and as an abrasive. With respect to DNA, diatomaceous earth has been used to separate cell wall fragments of Streptococcal cell lysates from which bacteriophages are prepared (Nugent et al., 1977, J. Virol. 21:1061-1073). No adsorption and release of DNA was involved. It has also been used in "nucleoprotein-Celite chromatography", which uses lysed nuclei adsorbed to Celite.RTM. (a commercial form of diatomaceous earth) and denaturing conditions and temperature to release the cellular DNA from the protein (S'Yakste et al., 1981. Mol. Biol. (Mosc.) 15:1321-1329; 1985, Mol. Biol. (Mosc.) 19:1231-1241). The protein itself is irreversibly adsorbed onto the Celite, while the nucleic acids do not interact with the Celite.
Thus, a method was still desired to rapidly and inexpensively separate and purify DNA that was also amenable to scale-up.